Method for determining gas hold-up

ABSTRACT

A method and apparatus for determining gas hold-up in a fermentation culture, in which, prior to aeration and during fermentation, a high frequency alternating signal is input to electrodes in electrical contact with the culture, the magnitude of the reactive current signal leading the voltage in the electrode circuit is determined and the difference in said magnitudes prior to aeration and during fermentation is used as a measure of the gas hold-up. The system may be combined with various methods of measuring biomass to give a gas hold-up corrected value of biomass.

This invention relates to determination of gas hold-up in a fermentationprocess and has wider implications for determination of cellular biomassor biovolume and to processes in which such determination is made.

One of the most important variables in a fermentation or other processusing biological cells is the reactor biomass concentration, that is theconcentration of microbial or other biological cells in the reactor,because the productivity of a process under a given set of conditions isproportional to the biomass concentration.

For monitoring purposes during a fermentation it is particularly usefulto be able to estimate the biomass on a real time basis rather than atsome time in the past. This generally requires the utilisation ofphysical properties that can be measured in situ rather than propertieswhich require sampling and analysis. However, the conditions withinfermentation reactors do not lend themselves to the use of all physicaltechniques.

In prior artwork done by the Applicant European Patent No. 0281602 andpublished European Patent Application No. 0282532 describe a method andapparatus for the determination of biomass in a culture medium in whicha signal dependent on the electrical capacitance or dielectricpermittivity is generated, at a suitable frequency or range offrequencies, between electrodes mutually spaced in the medium, anddetermining from the capacitance or permittivity dependent signal, thevolume of total liquid in a net volume fraction enclosed by thecytoplasmic membranes of the cells.

The basis of this approach is that biological cells, in contrast tomacromolecules, ionic solutions and gas bubbles, have molecularly thinlipid membranes which (when measured at suitable frequencies) can beshown to have a large electric capacitance per unit membrane area. Whensuspended in a conductive medium, the measured capacitance exhibits afrequency dependence known as the beta dispersion. By measuring thecapacitance at suitable frequencies it is possible to estimate themagnitude of the beta dispersion and in turn the biomass concentration.

In a development of the above approach a conductance signalrepresentative of the culture conductivity at respective high and lowfrequencies may also be used.

These methods work in many processes, but a problem that limits theiraccuracy is the varying quantity of gas that may be present in theculture. During fermentation the medium in a bioreactor may consist ofmultiple phases o gases, liquids and solids, and because thepermittivity of gases is significantly lower than that of liquids, thepresence of gas bubbles decreases the measured capacitance orconductance of the suspension causing errors in the biomassconcentration estimate. This problem can be minimized by maintaining aconstant level of aeration during the reaction and calibrating theapparatus for that level. However in some circumstances it may not beconvenient or possible to keep gas hold-up constant even when aerationrate is constant and it is desirable to have a measuring technique forgas hold-up.

Accordingly the invention provides a method of determining gas hold-upin a fermentation culture, the method comprising, prior to aeration andduring fermentation, inputting a high frequency alternating signal toelectrodes in electrical contact with the culture, determining themagnitude of the current signal leading the voltage in the electrodecircuit and using the difference in said magnitudes prior to aerationand during fermentation as a measure of the gas hold-up.

The invention also provides apparatus for determining gas hold-up in afermentation culture, the apparatus comprising means for inputting ahigh frequency alternating signal to an electrode circuit that includeselectrodes in electrical contact with the culture, means for inputtingthe voltage over the inter electrode gap to a phase sensitive detector,means for imposing a 90° phase lag on a current signal from theelectrode circuit and for inputting said lagged signal to the phasesensitive detector to produce a signal related to a quadrature componentof current in the electrode circuit.

The invention is now described by way of example with reference to theaccompanying drawings in which:

FIG. 1 illustrates a circuit for combined conductance measurement ofbiomass and gas hold-up correction;

FIG. 2 schematically illustrates a fermentation reactor provided withthe equipment of FIG. 1.

FIG. 3 schematically illustrates an alternative circuit using a separatefrequency for gas hold-up measurement; and

FIG. 4 schematically illustrates a circuit in which frequency signalsare applied sequentially rather than simultaneously.

As mentioned above, the electrical behaviour of a cellular suspension ortissue is strongly dependent on frequency, the frequency dependentchange in characteristic relative to the value for an aqueous solutionbeing termed a `dispersion`. Three major sources of dispersion arerecognised and are termed alpha, beta and gamma dispersion. Betadispersion, as explained in our prior European Patent No. 0281602, is aproperty of intact cells and is therefore of use in analysing cellcontent via the electrical behaviour of media incorporating intactbiological cells. Alpha and gamma dispersions are not predictablydependent upon intact cell concentration, and occur, respectively, atlower and higher frequencies than beta dispersion being related tomobile ions at cell surfaces and dipolar rotation.

Beta dispersion is essentially due to intact cells having a poorlyconducting, relatively ion-impermeable, cellular membrane, whereas theinterior of the cell is relatively conducting. The position of the betadispersion curve on the frequency axis is a function of cell radius,membrane capacitance and internal and external electricalconductivities, whilst the magnitude of the dispersion depends upon theconcentration, in terms of percentage volume, of the cells in the mediumas well as other variables such as cell size.

Our previous patent utilises a calibrated measurement of capacitance inthe frequency range of the beta dispersion as a measure of biovolume.However as measurement of capacitance in higher conductance mediabecomes more difficult, an alternative technique utilising conductancemeasurements has been developed. This alternative technique utilises thefact that at lower frequencies cell membranes block the flow of currentthrough the interior of the cell, but at higher frequencies currentflows through the cell via the membrane capacitance. It can be shownthat the difference in conductivity through the suspension at differentfrequencies equals 9/4 (volume fraction×internal cell conductivity),i.e. it depends upon, inter alia, the radius and volume fractionoccupied by the cells.

During fermentation the liquid media in reactors are aerated andalthough the rate of aeration can be maintained constant so as tomaintain, as far as possible, constant gas hold-up, there are occasionswhen changes in the rate of aeration are desired. Also, as fermentationprogresses changes in viscosity may also result, giving rise to changesin gas hold-up.

The presence of gas bubbles, reducing the unit volume concentration ofwater, reduces the capacitance over the whole range of frequencies usedin biomass measurement because of the lower permittivity of gas comparedwith water. The change in capacitance caused by gas hold-up has aneffect both on background capacitance and also upon the measuredmagnitude of the β dispersion itself because the gas bubbles alsodisplace cells. Hence in the capacitance measurement technique themeasured β dispersion changes with gas hold-up.

In the above described conductance method water displacement alsochanges the measured values. In this technique the difference betweentwo measurements is taken, so the background element of the change maybeeliminated, but this still leaves the inaccurancy in measured dispersionarising from displacement of the cells.

The present invention is principally concerned with enabling measurementof gas hold-up in order to establish corrected values for biomass(derived from biovolume) as measured for example by either of thetechniques referred to above.

At high frequencies, ideally above the β dispersion range, capacitanceis principally determined by water content. Thus in the invention it isproposed to utilise a high frequency capacitance measurement beforeonset of aeration, and during reaction. Subsequent changes in thecapacitance at the higher frequency after onset of aeration and reactionare then attributed directly to gas hold-up, for example a 20% reductionin capacitance indicating approximately 20% gas content.

This estimate of gas hold-up can be used to correct the biomassconcentration estimate provided by means such as the capacitance methodin EP 0281602 or, as described hereinafter, the conductance method. Ineither instance the following relationship applies: ##EQU1##

Conventional sampling followed by laboratory analysis methods, i.e.after the gas bubbles have cleared from the sample, measure correctedbiomass and thus the present invention enables in-situ determination ofthe conventional analysis amounts, which may be subsequently verifiedfrom time to time.

Preferably the frequency used for the capacitance measurement is abovethe β dispersion range. In some instances measurements may need to betaken within the top end of the β dispersion range, for example if thedispersion range is very extended. This would cause some inaccurancy ofthe gas fraction estimate; if this is unacceptably large, a curvefitting technique may be used to determine the true high-frequency (i.e.above β dispersion) capacitance value.

A measure of gas fraction, as opposed to just aeration rate, is alsouseful in its own right as a means of monitoring and assisting in theoptimisation of some fermentations.

A circuit for a preferred embodiment of the invention is shown in FIG.1, in which a gas hold-up measurement is combined with conductancemeasurement to determine biomass. The conductance measurement utilisesmeasurement at two frequencies generated by oscillators 1 and 2, withoscillator 2 at high frequency. The outputs of the oscillators arecombined, amplified, input to an automatic level control 3 and then tothe primary winding of an isolating transformer 4. Induced oscillationsoccur in an electrode circuit 5 connected to the transformer secondarywinding.

To alleviate electrode polarisation effects there are two sets ofelectrodes, outer current electrodes 7, 7' through which the currentpasses to and from the culture and at which any polarisation effectswill occur, and an inner set of voltage electrodes 8, 8' which samplethe voltage over a gap intermediate the current electrodes, immune frompolarisation. A separate circuit 6 for applying pulses to the electrodesfor cleaning purposes from time to time may also be provided. Otherelectrode configurations may also be used.

The voltages sensed by the electrodes 8, 8' are amplified and input to adetector 9 and to band-pass filters 10 and 11. Detector 9 completes afeedback loop for automatically controlling the gain of the appliedvoltage by means of the automatic level control 3. It will beappreciated that the voltage over the electrodes 8, 8' is a combinationof voltages having the two different oscillator frequencies and theband-pass filters 10 and 11 are tuned, respectively, to pass the lowerand higher frequency voltage signals which constitute phase referencesignals for the phase detectors 12 and 13.

From the band-pass filters the voltage signals are input to respectivephase sensitive detectors 12 and 13 which also receive a current signalinput from the electrode circuit, which for example may be taken over asuitable load. The output from the phase sensitive detectors 12 and 13are signals corresponding to the magnitude of the component of thecurrent signal that is in phase with the respective reference voltagesignal, i.e. proportional to the conductance. Thus conductance valuesignals for the lower and higher frequencies are output, respectively,from phase sensitive detectors 12, 13 into a comparator within adifference and correction circuit 14.

The circuit 14 also receives a temperature dependent signal from atemperature sensor 15 located close to the electrodes in the mediumwhich provides a temperature compensation adjustment from aprecalibrated reference.

The gas hold-up correction is also input to circuit 14. This utilisesthe higher frequency voltage signal, taken after band-pass filter 11 andinput to a third phase sensitive detector 16, along with the currentsignal. In order to establish the extent of gas hold-up a measure of thecapacitance at the higher frequency is required, and thus prior to thephase sensitive device the current signal is input to a phase shifter17, which imposes a π/2 lag, so that the output of phase sensitivedetector 16 is indicative of the magnitude of the current signal leadingby π/2 with respect to the voltage in the electrode circuit, i.e.representative of the capacitance value. This capacitance value is usedby the correction circuit, using pre-calibrated values, to apply a gashold-up correction to the final biomass concentration dependent signaloutput from circuit 14.

It will be realised that if the different frequency signals are appliedin rapid succession, rather than simultaneously, signal processing ofthe different frequencies may be handled within a single processor; forexample the signals may all go in sequence to a single phase sensitivedevice. This approach reduces the requirement for thisfrequency-selective filtering and may be preferred for this reason. Asuitable circuit is shown in FIG. 4, using only a single oscillator 1, asequencing circuit 40 and with a sample-hold facility in the differenceand correction circuits 14.

FIG. 2 schematically illustrates a fermentation reactor 20 provided withconductance biovolume measuring equipment as previously described. Inorder to reduce equipment introduced errors the electrode voltage andcurrent sensing circuits including an amplification stage are preferablyincorporated in a probe module 21 close to the electrode circuit. Themain electronic processing unit 22 may then be located remote from theapparatus.

For simplicity the apparatus and method for combined biomass measurementand gas hold-up correction have been described in terms of utilising ahigh and low frequency. More than one high and low frequency may beutilised and the results used to compute biomass concentration and otherparameters or used for other correction purposes. In some cases, the gashold-up capacitance is preferably measured at a third, higher frequency,than the conductance measurement frequencies with suitable additionalcircuitry as shown in FIG. 3 with oscillator 30. Alternatively, the highfrequency capacitance measurement may be incorporated into a capacitancemeasuring biomass arrangement to correct the biomass concentrationsignal for changes in gas hold up. In some instances measurements mayneed to be taken within the top end of the β dispersion range ratherthan clear of it, for example if the dispersion range is very extended.In such instances, as with the conductance method above, it is possibleto utilise either separate or common high frequency signals for thebiomass concentration and gas hold-up measurement.

Also, different electrodes may be used for the gas hold-up measurementand biomass measurements.

The block diagrams shown herein have been simplified for illustrativepurposes. Production versions of the systems shown would require furtherfeedback systems for automatic trimming of zero level drift and phaseresponse of the electronics and cables.

I claim:
 1. A method of determining gas hold-up in a fermentationculture having a concentration of biological cells in a biomass, themethod comprising the steps of first prior to aeration and then duringfermentation of the culture inputting inputting to the cultureelectrically coupling an alternating signal of a frequency which isabove the range of frequencies at which β dispersion occurs, determiningthe magnitude of reactive current signal leading voltage in the cultureand using measured difference in said magnitudes determined prior toaeration and determined during fermentation as a measure of the gashold-up.
 2. The method of claim 1 further comprising determiningbiovolume in a culture and utilizing said gas hold-up determination toprovide a corrected biovolume estimate.
 3. The method of claim 2 inwhich the biovolume is determined by inputting respective high and lowfrequency alternating signals to the culture, deriving a signalrepresentative of a characteristic of the culture at each frequency andprocessing said signals to provide an output indicative of thebiovolume.
 4. The method of claim 3 in which the characteristic isconductance and the signals are generated by determining for eachfrequency a signal related to the in-phase component of current withinthe culture.
 5. The method of claim 2 in which the biovolume isdetermined via a capacitance measurement.
 6. The method of claim 2 inwhich the biovolume determination is subjected to a temperaturedependent adjustment.
 7. The method of claim 3 in which the gas hold-updetermination alternating frequency signal is one of the signals usedfor deriving biovolume.
 8. The method of claim 3 in which the gashold-up determination alternating frequency signal is a separate highfrequency signal not used for deriving biovolume.
 9. The method of claim3 in which at least some of the differing frequency signals are input tothe culture simultaneously.
 10. The method of claim 3 in which at leastsome of the differing frequency signals are input to the culturesequentially.
 11. The method of claim 1 in which the signals are inputby induction electrically to the culture.
 12. A method of determininggas hold-up in a fermentation culture having a concentration ofbiological cells in a biomass, the method comprising, prior to aerationand then during fermentation of the culture, inputting inputting to theculture electrically coupling an alternating signal at a frequency whichis within an upper part of the range of frequencies at which βdispersion occurs, determining the magnitude of reactive current signalleading voltage in the culture and using measured difference in saidmagnitudes determined prior to aeration and determined duringfermentation as a measure of the gas hold-up.
 13. The method of claim 12in which a curve fitting technique is used to determined a reactivecurrent signal value above the β dispersion range.
 14. The method ofclaim 12 further comprising determining biovolume in a culture andutilizing said gas hold-up determination to provide a correctedbiovolume estimate.
 15. The method of claim 14 in which the biovolume isdetermined by inputting respective high and low frequency alternatingsignals to the culture, deriving a signal representative of acharacteristic of the culture at each frequency and processing saidsignals to provide an output indicative of the biovolume.
 16. The methodof claim 15 in which the characteristic is conductance and the signalsare generated by determining for each frequency a signal related to thein-phase component of current within the culture.
 17. The method ofclaim 14 in which the biovolume is determined via a capacitancemeasurement capacitance.
 18. The method of claim 14 in which thebiovolume determination is subjected to a temperature dependentadjustment.
 19. The method of claim 15 in which the gas hold-updetermination alternating frequency signal is one of the signals usedfor deriving biovolume.
 20. The method of claim 15 in which the gashold-up determination alternating frequency signal is a separate highfrequency signal not used for deriving biovolume.
 21. The method ofclaim 15 in which at least some of the differing frequency signals areinput to the culture simultaneously.
 22. The method of claim 15 in whichat least some of the differing frequency signals are input to theculture sequentially.
 23. The method of claim 12 in which signals areinput by induction electrically to the culture.
 24. Apparatus fordetermining gas hold-up in a fermentation culture including biologicalcells, the apparatus comprising a probe circuit electrically coupledmeans for inputting to the culture for inputting a high frequencyalternating signal at least in the upper portion of the β dispersionrange, means for inputting a signal representative of the voltage overthe culture to a phase sensitive director, means for imposing a 90°phase lag on a signal representative of current through the culture andfor inputting said lagged signal to the phase sensitive detector toproduce a signal related to a quadrature component of current in theculture and a difference circuit for receiving a signal related to thequadrature component of current before onset of aeration of the cultureand subsequently receiving a signal related to the quadrature componentduring fermentation and establishing a difference value which is outputas a measure of gas hold-up.
 25. The apparatus of claim 24 in which saidsignal is at a frequency above the β dispersion range.
 26. The apparatusof claim 24 further comprising means for determining biovolumecomprising means for inputting respective high and low frequencyalternating signals to the culture and means for generating from therelative magnitude of characteristics of at least one of current andvoltage at the respective high and low frequencies an output signalindicative of biovolume.
 27. The apparatus of claim 24 in which thealternating signals are input to the culture by induction electricallyto the culture via probe circuit.
 28. The apparatus of claim 26 in whichthe means for generating comprises band-pass filters tuned to arespective one of the frequencies and connected to means for obtainingan in-phase component of current signal relative to the respectivevoltage signal, the outputs of which are input to a difference circuit.29. The apparatus of claim 28 including means for sampling voltagesignals over the culture and means for determining current signals inthe culture, the voltage signals being input to the band-pass filtersand the current signals input, along with the output from the band-passfilters to the means for obtaining the in-phase component.
 30. Theapparatus of claim 26 further comprising means for adjusting the outputsignal indicative of biovolume in dependence upon temperature sensed inthe culture.
 31. The apparatus of claim 26 further comprising means foradjusting the output signal indicative of biovolume in dependence uponthe magnitude of said difference value.
 32. Apparatus for determininggas hold-up and providing a corrected biovolume estimate for aconcentration of biological cells in a biomass, the apparatuscomprising:means for measuring biovolume comprising means for inputtingto the culture via electrode probes electrically coupled to the culturealternating signals at spaced apart frequencies within the β dispersionrange and means for generating from the relative magnitude of resultingcharacteristics of at least one of current and voltage at said spacedapart frequencies an output signal indicative of biovolume; and meansfor correcting for gas hold-up said output signal indicative ofbiovolume, said means for correcting comprising means for inputting tothe culture a high frequency alternating signal at a frequency at leastin the upper part of the β dispersion range and means for generatingfrom the relative magnitude of quadrature components of current withinthe culture resulting from input of said high frequency signal prior toaeration of the culture and subsequently input during aeration adifference frequency value of alternating signal, and means foradjusting said output signal indicative of biovolume in dependence uponthe magnitude of said difference frequency value of quadrature currentcomponent.
 33. The apparatus of claim 32 in which the high frequencyalternating signal is at a frequency above the β dispersion range. 34.The apparatus of claim 32 in which said high frequency signal is alsoone of said alternating signals at spaced apart frequencies.
 35. Theapparatus of claim 32 in which at least some of said high frequencysignal and signals at spaced apart frequencies are input to the culturesimultaneously.
 36. The apparatus of claim 32 in which at least some ofsaid high frequency signal and signals at spaced apart frequencies areinput to the culture serially.
 37. The apparatus of claim 32 furthercomprising curve fitting means for adjusting said quadrature componentof current to a value such that the alternating frequency of the currentis above the β dispersion range.
 38. The apparatus of claim 24 furthercomprising curve fitting means for adjusting said quadrature componentof current to a value such that the alternating frequency of the currentis above the β dispersion range.